Methods for the preservation of living biological materials are employed in many clinical and veterinary applications wherein living material, including cells, tissues and organs, is harvested and stored in vitro for some period of time before use. Examples of such applications include whole blood transplants, platelet transplants, autologous and allogeneic bone marrow transplants, organ storage and transplants, embryo transfer, artificial insemination, in vitro fertilization, skin grafting and storage of tissue biopsies for diagnostic purposes. Preservation techniques are also important in the storage of cell lines for experimental use in hospital, industrial, university and other research laboratories.
Methods currently employed for the preservation of cellular biological materials include immersion in saline-based media; storage at temperatures slightly above freezing; storage at temperatures of about -80.degree. C.; storage in liquid nitrogen at temperatures of about -196.degree. C.; and freeze-drying or lyophilization. The goal of all these techniques is to store living biological materials for an extended period of time with minimal loss of normal biological structure and function.
Saline-based media employed in the preservation of living biological materials typically consist of isotonic saline (sodium chloride 0.154 M) which has been modified by the addition of low concentrations of various inorganic ions such as--potassium, calcium, magnesium, chloride, phosphate and bicarbonate to mimic the extracellular environment. Small amounts of compounds such as glucose, amino acids and vitamins are often added as metabolites. Examples of media currently employed for the preservation of biological materials include phosphate-buffered saline (PBS), M-2 (a Hepes buffered murine culture medium), Ringer's solution and Krebs bicarbonate-buffered medium. The viability of biological materials stored in saline-based media above 0.degree. C. gradually decreases over time; living tissues can only be successfully preserved for relatively short periods of time.
When employing freezing techniques to preserve biological materials, high concentrations (approximately 10% by volume) of cryoprotectants, such as glycerol, dimethylsulfoxide (DMSO), glycols or propanediol, are often introduced to the material prior to freezing in order to limit the amount of damage caused to cells by the formation of ice crystals during freezing. The choice and concentration of cryoprotectant, time-course for the addition of cryoprotectant and temperature at which the cryoprotectant is introduced all play an important role in the success of the preservation procedure. Furthermore, in order to reduce the loss of cells, it is critical that such variables as the rate and time-course of freezing, rate and time-course of thawing and further warming to room or body temperature, and replacement of cryoprotectant solution in the tissue mass with a physiological saline solution be carefully controlled. The large number of handling steps required in freezing techniques increases the loss of cells. The freezing techniques currently employed in the preservation of biological materials are both technically demanding and time consuming. Other disadvantages of preserving biological materials by conventional freezing methods include: reduction of cell viability; toxic effects of the cryoprotectant to the patient upon re-infusion; the high costs of processing and storage; and the difficulty of transporting frozen materials.
For example, the use of conventional freezing methods in the preservation of platelets results in a progressive deterioration in cell function to such a degree that platelets are typically stored at room temperature. However, due to the risk of bacterial contamination, platelet storage at room temperature is generally restricted to five days. As a result, approximately 20% of transfusable platelet units must be discarded.
The high costs associated with maintaining preserved biological materials at freezing temperatures, together with the problems associated with transporting frozen materials, can be avoided by lyophilization, or freeze-drying. Lyophilized materials can be stored at room temperature for extended periods of time and then readily reconstituted for use. However, while proteins have been successfully preserved by lyophilization, this technique has to date been of limited use in the preservation of whole cells.
U.S. Pat. No. 5,242,792 describes a method for the lyophilization of red blood cells which includes contacting the cells with a protective agent selected from the group consisting of either sucrose, raffinose, maltose, lactose or trehalose in a buffered solution and permeabilizing the cells by contacting them with either inositol or glycerol prior to freezing and lyophilizing. International Patent Application No. WO 93/00807 discloses an additive for stabilizing biological materials during lyophilization including a cryoprotectant, such as polyethylene glycol, and a second component such as a sugar, polyhydroxyl alcohol, amino acid or methylamine. U.S. Pat. No. 5,045,446 teaches a method for lyophilizing cells which employs a solution comprising monosaccharide hexoses and pentoses, and a mixture of at least two amphipathic polymers. International Patent Application No. WO 93/14191 teaches a process for freezing or freeze-drying cells which uses a cryoprotectant medium having an elevated glass transition temperature.